The role of the anion-binding site of transferrin in its interaction with the reticulocyte

Specific and tight binding of Fe(III) by transferrin does not occur unless a suitable anion is concomitantly bound. Bicarbonate, which normally occupies the anion binding site of the protein, may be replaced by an oxalate ion. The resulting ternary complex of Fe(III), transferrin and oxalate is less than 35% as effective as the bicarbonate complex in serving as an iron donor for heme synthesis by the reticulocyte. However, the binding of transferrin to the reticulocyte is not altered by the substitution of oxalate for bicarbonate. When both the oxalate and bicarbonate forms are incubated with reticulocytes, the uptake of iron from the bicarbonate complex is substantially depressed. The free oxalate ion, at the same concentration as the ternary Fe-transferrin-oxalate complex, does not alter the uptake of iron by reticulocytes from the native form of transferrin. The ternary Fe-transferrin-malonate complex is also less efficient than the bicarbonate complex as an iron donor to the reticulocyte, but the effect is less striking than that observed with the oxalate complex. The hypothesis is advanced that the mechanism of iron uptake from transferrin during the transferrin-reticulocyte interaction first entails an attack upon the anion bound to the protein, following which iron release to the heme-synthesizing apparatus of the cell takes place.     

Crystal structures and iron release properties of mutants (K206A and K296A) that abolish the dilysine interaction in the N-lobe of human transferrin

Human transferrin (Tf) is responsible for the binding and transport of iron in the bloodstream of vertebrates. Delivery of this bound iron to cells occurs by a process of receptor-mediated endocytosis during which Tf releases its iron at the reduced endosomal pH of approximately 5.6. Iron release from Tf involves a large conformational change in which the two domains that enclose the binding site in each lobe move apart. We have examined the role of two lysines, Lys206 and Lys296, that form a hydrogen-bonded pair close to the N-lobe binding site of human Tf and have been proposed to form a pH-sensitive trigger for iron release. We report high-resolution crystal structures for the K206A and K296A mutants of the N-lobe half-molecule of Tf, hTf/2N, and quantitative iron release data on these mutants and the double mutant K206A/K296A. The refined crystal structures (for K206A, R = 19.6% and R(free) = 23.7%; for K296A, R= 21.2% and R(free) = 29.5%) reveal a highly conserved hydrogen bonding network in the dilysine pair region that appears to be maintained even when individual hydrogen bonding groups change. The iron release data show that the mutants retain iron to a pH 1 unit lower than the pH limit of wild type hTf/2N, and release iron much more slowly as a result of the loss of the dilysine interaction. Added chloride ions are shown to accelerate iron release close to the pH at which iron is naturally lost and the closed structure becomes destabilized, and to retard it at higher pH.     

Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296.

In previous studies, mutation of either Lys296 or Glu113 in bovine rhodopsin has been shown to result in constitutive activation of the apoprotein form, opsin [Robinson et al. (1992) Neuron 9, 719-725]. In this report, pH-rate profiles for the rhodopsin-catalyzed exchange of GTPgS for GDP on transducin are established for the constitutively active opsin mutants. All of the mutants, including the double-mutant E113Q,K296G, show a bell-shaped pH-rate profile. Therefore, it is evident that at least two ionizable groups in addition to Lys296 and Glu113 control the formation of the active opsin state. The sole effect of mutation at position 113 or 296 is to alter the ionization constant of the group with the higher pKa, called pka2. pKa2 decreases in the following order: rhodopsin/light (9.0) > K296E = K296G = E113Q,K296G (8.0) > E113Q (6.8) > K296H (6.6) > wild-type opsin (< 5.0). These results are consistent with a model where activation of opsin involves (i) breaking of the salt bridge between Lys296 and Glu113, (ii) deprotonation of Lys296, and (iii) the net uptake of a proton from the solvent. Furthermore, exogenous addition of the chromophore all-trans-retinal shifts the wild-type and E113Q opsin equilibrium to favor the active state. In all these respects, the light-independent activation of the opsin mutants appears to proceed by a mechanism similar to that of light-activated rhodopsin.     

Structural and functional consequences of binding site mutations in transferrin: crystal structures of the Asp63Glu and Arg124Ala mutants of the N-lobe of human transferrin

Human transferrin is a serum protein whose function is to bind Fe(3+) with very high affinity and transport it to cells, for delivery by receptor-mediated endocytosis. Structurally, the transferrin molecule is folded into two globular lobes, representing its N-terminal and C-terminal halves, with each lobe possessing a high-affinity iron binding site, in a cleft between two domains. Central to function is a highly conserved set of iron ligands, including an aspartate residue (Asp63 in the N-lobe) that also hydrogen bonds between the two domains and an arginine residue (Arg124 in the N-lobe) that binds an iron-bound carbonate ion. To further probe the roles of these residues, we have determined the crystal structures of the D63E and R124A mutants of the N-terminal half-molecule of human transferrin. The structure of the D63E mutant, determined at 1.9 A resolution (R = 0.245, R(free) = 0.261), showed that the carboxyl group still binds to iron despite the larger size of the Glu side chain, with some slight rearrangement of the first turn of alpha-helix residues 63-72, to which it is attached. The structure of the R124A mutant, determined at 2.4 A resolution (R = 0.219, R(free) = 0.288), shows that the loss of the arginine side chain results in a 0.3 A displacement of the carbonate ion, and an accompanying movement of the iron atom. In both mutants, the iron coordination is changed slightly, the principal change being in each case a lengthening of the Fe-N(His249) bond. Both mutants also release iron more readily than the wild type, kinetically and in terms of acid lability of iron binding. We attribute this to more facile protonation of the synergistically bound carbonate ion, in the case of R124A, and to strain resulting from the accommodation of the larger Glu side chain, in the case of D63E. In both cases, the weakened Fe-N(His) bond may also contribute, consistent with protonation of the His ligand being an early intermediate step in iron release, following the protonation of the carbonate ion.     

The synergistic anion-binding sites of human transferrin: chemical and physiological effects of site-directed mutagenesis

A defining feature of all transferrins is the absolute dependence of iron binding on the concomitant binding of a synergistic anion, normally but not necessarily carbonate. Acting as a bridging ligand between iron and protein, it completes the coordination requirements of iron to lock the essential metal in its binding site. To investigate the role of the synergistic anion in the iron-binding and iron-donating properties of human transferrin, a bilobal protein with an iron binding site in each lobe, we have selectively mutated the anion-binding threonine and arginine ligands that form an essential part of the electrostatic and hydrogen-bonding network holding the synergistic anion to the protein. Preservation of either ligand is sufficient to maintain anion binding, and therefore iron binding, in the mutated lobe. Arginine is a stronger ligand than threonine, and its loss weakens carbonate and therefore iron binding, but maintains the ability of nitrilotriacetate to serve as a carbonate surrogate. Replacement of both ligands abolishes anion binding and consequently iron binding in the affected lobe. Loss of anion binding in either lobe results in a monoferric protein binding iron in normal fashion only in the opposite lobe. Both monoferric proteins are capable of transferrin receptor-dependent binding and iron donation to K562 cells, but with diminished receptor occupancy by the protein bearing iron only in the N-lobe.     

Crystal structures of two mutants (K206Q, H207E) of the N-lobe of human transferrin with increased affinity for iron

The X-ray crystallographic structures of two mutants (K206Q and H207E) of the N-lobe of human transferrin (hTF/2N) have been determined to high resolution (1.8 and 2.0 A, respectively). Both mutant proteins bind iron with greater affinity than native hTF/2N. The structures of the K206Q and H207E mutants show interactions (both H-bonding and electrostatic) that stabilize the interaction of Lys296 in the closed conformation, thereby stabilizing the iron bound forms.     

Anion exchange in human serum transferrin N-lobe: a model study with variant His249Ala

The removal of Fe(III) from human serum transferrin by chelators is thought to proceed through intermediate species in which the chelator becomes associated with the metal center of the protein. The visible spectral shifts associated with the formation of such intermediates in the wild-type (WT) protein are too small for reliable kinetic data to be obtained. Therefore, studies were undertaken with the recombinant N-terminal lobe variant H249A, a variant showing more pronounced spectral changes. The kinetics of the synergistic anion-exchange reaction between nitrilotriacetate (NTA) and carbonate in variant H249A was studied by stopped-flow spectrophotometry as a model for this process in the WT protein. Anion exchange occurs by two pathways at pH 7.4 and 25 degrees C: an NTA-independent dissociative pathway to form a carbonate-free intermediate Fe-H249A (Eq. 1) that subsequently reacts with NTA (Eq. 2):and an NTA-dependent associative pathway (the major pathway) in which a quaternary Fe-H249A-(CO(3))(NTA) intermediate is formed (Eq. 3), which then decays to product (Eq. 4):The reverse reaction, where HCO(3)(-) exchanges for NTA, likewise follows these two pathways. The overall apparent equilibrium constant for formation of Fe-H249A-NTA from Fe-H249A-CO(3) is K'=442 at pH 7.4. The NTA complex is favored over the carbonate complex both kinetically and thermodynamically in the pH range 7.4-8.2.     

Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg(47)Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg(47)Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg(47)Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro(53)Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu(49) and Leu(56) were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys(55), which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.     

A computational study of the open and closed forms of the N-lobe human serum transferrin apoprotein

Human serum transferrin tightly binds ferric ions in the blood stream but is able to release them in cells by a process involving receptor-mediated endocytosis and decrease in pH. Iron binding and release are accompanied by a large conformation change. In this study, we investigate theoretically the open and closed forms of the N-lobe human serum transferrin apoprotein by performing pKa calculations and molecular dynamics and free-energy simulations. In agreement with the hypothesis based on the x-ray crystal structures, our calculations show that there is a shift in the pKa values of the lysines forming the dilysine trigger when the conformation changes. We argue, however, that simple electrostatic repulsion between the lysines is not sufficient to trigger domain opening and, instead, propose an alternative explanation for the dilysine-trigger effect. Analysis of the molecular dynamics and free-energy results indicate that the open form is more mobile than the closed form and is much more stable at pH 5.3, in large part due to entropic effects. Despite a lower free energy, the dynamics simulation of the open form shows that it is flexible enough to sample conformations that are consistent with iron binding.     

Identification of possible kinetically significant anion-binding sites in human serum transferrin using molecular modeling strategies

Certain anions have been shown experimentally to influence the rate of iron release from human serum transferrin (HST), implying the existence of one or more allosteric kinetically significant anion-binding (KISAB) sites on or near the surface of the protein. A rank-ordered selection of potential HST KISAB sites has been obtained using a novel three-stage molecular modeling strategy. The crystal structure of HST (1A8E.pdb) was first subjected to a heuristic analysis, in which positively charged and hydrogen-bonding residues on or near the surface of the protein were identified. In this stage, a preliminary electrostatic potential map was also calculated, yielding six preliminary sites. Next, energy-grid calculations were conducted in order to identify anion-protein interaction energy minima, which resulted in the inclusion of three additional sites. Finally, three anions already shown experimentally to demonstrate varied effects on HST iron-release kinetics were placed at each potential site; molecular dynamics and molecular mechanics calculations were performed in order to elucidate the hydrogen-bonding environment around each anion of the protein as well as to calculate anion-protein-binding energies.     

Allosteric changes of thrombin catalytic site induced by interaction of bothrojaracin with anion-binding exosites I and II.

Bothrojaracin, a 27-kDa C-type lectin from Bothrops jararaca venom, is a selective and potent thrombin inhibitor (K(d) = 0.6 nM) which interacts with the two thrombin anion-binding exosites (I and II) but not with its catalytic site. In the present study, we analyzed the allosteric effects produced in the catalytic site by bothrojaracin binding to thrombin exosites. Opposite effects were observed with alpha-thrombin, which possesses both exosites I and II, and with gamma-thrombin, which lacks exosite I. On the one hand, bothrojaracin altered both kinetic parameters K(m) and k(cat) of alpha-thrombin for small synthetic substrates, resulting in an increased efficiency of alpha-thrombin catalytic activity. This effect was similar to that produced by hirugen, a peptide based on the C-terminal hirudin sequence (residues 54-65) which interacts exclusively with exosite I. On the other hand, bothrojaracin decreased the amidolytic activity of gamma-thrombin toward chromogenic substrates, although this effect was observed with higher concentrations of bothrojaracin than those used with alpha-thrombin. In agreement with these observaions, bothrojaracin produced opposite effects on the fluorescence intensity of alpha- and gamma-thrombin derivatives labeled at the active site with fluorescein-Phe-Pro-Arg-chloromethylketone. These observations support the conclusion that bothrojaracin binding to thrombin produces two different structural changes in its active site, depending on whether it interacts exclusively with exosite II, as seen with gamma-thrombin, or with exosite I (or both I and II) as observed with alpha-thrombin. The ability of bothrojaracin to evoke distinct modifications in the thrombin catalytic site environment when interacting with exosites I and II make this molecule an interesting tool for the study of allosteric changes in the thrombin molecule.     

Mutation of the iron ligand His 249 to Glu in the N-lobe of human transferrin abolishes the dilysine "trigger" but does not significantly affect iron release

Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive "trigger" involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half-molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8%, R(free) 29.4%), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site.     

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin.

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin has been studied by analyzing the distribution of iron between the chelator and the proteins as a function of both ligand concentration and transferrin saturation. The kinetics of iron removal by 3-hydroxypyridin-4-ones from both transferrins is slow; in ovotransferrin it appears to be monophasic, in contrast to that observed for serum transferrin. After 24 hours incubation at a 40:1 chelator:protein molar ratio, the percentage of iron removed from Fe(III)-ovotransferrin is 50%-60%, and is somewhat higher in the case of serum transferrin, in line with the respective affinity constants for the metal. The 3-hydroxypyridin-2-ones and the 3-hydroxypyran-4-ones, both of which have lower affinities for Fe(III), remove smaller proportions of the metal. The percentage of desaturation obtained with bidentate and hexadentate pyridinones appears to be similar for both transferrin classes at chelator:protein molar ratios from 40:1. The degree of transferrin saturation influences the extent of chelator mediated iron mobilization in the case of serum transferrin, but not of ovotransferrin. 59Fe competition studies demonstrate that bidentate pyridin-4-ones are capable of donating iron to serum apotransferrin; the relative concentrations of ligand and protein influence the distribution of iron because their effective binding constants (at pH 7.4) for Fe(III) are similar.     

Intrinsic fluorescence reports a global conformational change in the N-lobe of human serum transferrin following iron release

Transferrins have been extensively studied in order to understand how they reversibly bind and release iron. Human serum transferrin (hTF) is a single polypeptide chain that folds into two lobes (N- and C-lobe); each lobe binds a single ferric ion. Iron release induces a large conformational change in each lobe. At the putative endosomal pH of 5.6, measurement of the increase in intrinsic fluorescence upon iron release from the recombinant N-lobe yields two rate constants: 8.9 min-1 and 1.3 min-1. Direct monitoring of iron release from the N-lobe at pH 5.6 (by the decrease in absorbance at 470 nm) gives a single rate constant of 9.1 min-1, definitively establishing that the faster rate constant in the fluorescent studies is due to iron release. To further elucidate the molecular basis of the intrinsic fluorescence change (and the source of the slower rate constant), we examined the contributions of the three individual tryptophan residues in the N-lobe (Trp8, Trp128, and Trp264). Three double mutants, each containing the single remaining tryptophan residue, were produced. In the iron-bound N-lobe, Trp128 and Trp264 are quenched by iron and account for almost the entire fluorescent signal when iron is released. As for the wild-type N-lobe, the fluorescence increase for each of these mutants is best fit by a double-exponential function indicating two processes. Trp8 is severely quenched under all conditions, making virtually no contribution to the signal. Additionally, a mutant lacking all three Trp residues allows assignment of the fluorescent signal completely to the three tryptophan residues and observation of the presence of one (or more) tyrosinates in the N-lobe that have physiological significance in the uptake of iron.     

Histidine pK(a) values for the N-lobe of human transferrin: effect of substitution of binding site Asp by Ser (D63S)

The pK(a) values have been determined for eight of the nine histidine residues and the amino terminus of the N-lobe of human apo-transferrin (hTF/2N), and for seven of the nine histidine residues and the amino terminus of the protein Asp63Ser hTF/2N containing a mutation of the Fe(3+)-ligand Asp63 to Ser63. Calculations suggested that substitution of aspartate by serine would result in decreases of the pK(a) values of most of the histidine residues in the protein. This was found to be the case experimentally, and allowed assignment of the varepsilonCH resonance of His249. For the wild-type protein, the His residue with a pK(a) of 7.40 was assigned as His249, whereas for the mutant, no observable His residue had a pK(a) value higher than 6.9. The protonated form of His249 appears to be stabilised by interactions with Asp63, and the high pK(a) value may be critical for ensuring the release of iron at endosomal pH (5.5). The mutation lowered the apparent binding constant of hTF/2N for the synergistic anion oxalate from log K 4.0 to log K 3.3. (1)H NMR spectral changes induced by Ga(3+) binding to the mutant are compared to those observed for the wild-type protein.     

Localization of the single-stranded DNA binding site in the thrombin anion-binding exosite.

Single-stranded DNA molecules containing a 15-nucleotide consensus sequence have been reported to inhibit thrombin activity. The mechanism of the inhibition was studied using a consensus 15-mer oligonucleotide and two recombinant mutant thrombins: the anion-binding exosite mutant thrombin R70E, and thrombin K154A, in which the mutation was located in a surface loop outside of the exosite. The consensus 15-mer oligonucleotide inhibited both fibrinogen-clotting and platelet-activation activities of plasma-derived thrombin, recombinant wild type thrombin, and mutant thrombin K154A in a sequence-specific and dose-dependent manner, whereas it did not inhibit either activity of mutant thrombin R70E. The 15-mer oligonucleotide also inhibited thrombomodulin-dependent protein C activation by plasma-derived thrombin. In competition equilibrium binding experiments, binding of 125I-labeled diisopropyl phosphoryl-thrombin to thrombomodulin was completely inhibited by the consensus 15-mer oligonucleotide with a Kd value of 2.68 +/- 0.16 nM. These results suggest that Arg-70 in the anion-binding exosite of thrombin is a key determinant for interaction with specific single-stranded DNA molecules, and that binding of single-stranded DNA molecules to the exosite prevents the interaction of thrombin with fibrinogen, the platelet thrombin receptor, and thrombomodulin.     

Identification of the O-linked glycosylation site of the human transferrin receptor.

The human transferrin receptor is a glycoprotein containing three N-linked and one O-linked glycosylation sites. Tryptic digestion of the receptor, followed by chromatography on BioGel P-2 and reverse-phase HPLC, yields a glycopeptide (amino acids 101-120) containing the O-linked site. Amino acid sequence analysis reveals that the site of O-glycosylation is Thr-104. Mass spectral analysis is consistent with the presence of a Gal-GalNAc core with predominantly two sialic acid residues.